1. Field of the Invention
The present invention relates to inhibition of plant pathogens, and more specifically, to the increased expression of RPW8 with an increased levels at the extrahaustorial membrane and subsequent induction of pathogen inhibition reactions.
2. Background of Related Technology
Plant diseases caused by various pathogens pose a constant threat to human food security. Plant defense against invading obligate, biotrophic haustorium-forming fungal and oomycete pathogens occurs at pre-invasion and post-invasion stages (Lipka et al., 2005). Pre-invasion resistance protects plants from non-adapted pathogens, by blocking their entry into the host cell (Collins et al., 2003; Lipka et al., 2005; Stein et al., 2006). One common induced cellular defense response is the deposition of defense chemicals including callose (β-1,3-glucan) at the site of penetration, resulting in cell-wall apposition, a subcellular structure also known as a papilla (Aist, 1976; Huckelhoven et al., 1999).
Plants have evolved at least two distinct mechanisms for induction of pre-invasion resistance. The first engages the PEN1 syntaxin-containing SNARE (soluble N-ethylmalemide-sensitive factor attachment protein receptor) complex-dependent exocytosis pathway for delivering yet-to-be-defined antimicrobial materials to the site of penetration (Collins et al., 2003; Kwon et al., 2008). The second involves the accumulation of 4-methoxyindol-3-ylmethylglucosinolate (which leads to callose formation) through hydrolysis of indole glucosinolates by the PEN2 atypical myrosinase and action of the plasma membrane-localized PEN3 ATP-binding cassette transporter (Lipka et al., 2005; Stein et al., 2006; Bednarek et al., 2009; Clay et al., 2009). Loss-of-function of PEN1, PEN2 or PEN3 in Arabidopsis each results in higher penetration rates of non-adapted powdery mildew pathogens.
For an adapted pathogen, however, pre-invasion resistance is ineffective; the pathogen has evolved the ability to penetrate the host cell wall underneath the appressorium and produce a feeding structure, the haustorium, presumably through invagination of the host plasma membrane (PM). This interfacial membrane, termed extrahaustorial membrane (EHM) (Mackie et al., 1991), is poorly characterized, and to date not a single protein either from the host or the pathogen has been definitively identified as an EHM-resident. Apart from nutrient take-up from the host cell (Manners and Gay, 1982; Voegele et al., 2001), the haustorium functions to secrete effector proteins into the host cell across the interface to suppress host defenses including those elicited upon recognition of microbe-associated molecular patterns (MAMPs) by cell surface receptors (Catanzariti et al., 2007). The formation and sustained functioning of the haustorium enables successful colonization of the invading pathogen on the plant, and establishment of long-term parasitic relationship with the host.
Facing selective pressure imposed by the pathogens, plants have evolved post-invasion resistance mechanisms, often controlled by dominant resistance (R) genes, whose products directly or indirectly detect specific pathogen effectors and trigger effective defense responses (Jones and Dangl, 2006). R protein-triggered resistance to various pathogens is normally race-specific and only effective against pathogen strains expressing the cognate effector protein recognized by the R protein, and often associated with a hypersensitive response (HR), which is manifested as rapid death of the invaded cell and in some cases a few surrounding cells (Hammond-Kosack and Jones, 1997; Morel and Dangl, 1997; Schulze-Lefert and Panstruga, 2003). Most characterized plant R proteins belong to members of the nucleotide-binding-site, leucine-rich-repeat (NB-LRR) protein superfamily (Jones and Dangl, 2006). Several isolated powdery mildew resistance genes belong to this class. Those include the R genes at the Mla locus of barley (Halterman et al., 2001; Zhou et al., 2001; Halterman and Wise, 2004) and Pm3b of wheat (Yahiaoui et al., 2004).
Identification and utilization of naturally evolved plant disease resistance (R) genes provide the best way to control crop disease problems. Having been subject to the long-term “arms-race” between plants and pathogens, most R genes are highly specialized in activating resistance to only one or a few strains of a particular pathogen and they can be defeated by the faster-evolving pathogens in a short period of time. Durable and broad-spectrum R genes are thus among the most desirable for sustainable agriculture. The first durable and broad-spectrum R gene RPW8 (AF273059) isolated from Arabidopsis thaliana confers resistance to powdery mildew diseases caused by Erysiphe spp, an important group of fungal pathogens belonging to Ascomycete (S. Xiao, J. G. Turner, M. Coleman, and S. Ellwood, Plant resistance gene, PCT Application No. WO0198479, Plant Bioscience Limited (GB), UK).
The two homologous Arabidopsis R genes RPW8.1 and RPW8.2 from the Ms-0 accession confer broad-spectrum (Xiao et al., 2001) to host-adapted Golovinomyces spp. fungi belonging to Ascomycete, which are the causal agents of powdery mildew diseases on numerous dicot plant species (Braun, 1987). RPW8.1 and RPW8.2 encode proteins that contain an N-terminal transmembrane (TM) and 1-2 coiled-coil (CC) domains (Xiao et al., 2001), and thus are placed in an unusual class of R genes (Dangl and Jones, 2001). Despite their atypical predicted protein structure, RPW8.1 and RPW8.2 (both together are referred to as RPW8 unless otherwise indicated) activate HR and other defense responses via the conserved salicylic acid (SA)-dependent signaling pathway also recruited by a subset of NB-LRR R proteins that contain an N-terminal TIR (toll and interleukin-1 receptor) domain for race-specific resistance (Xiao et al., 2003; Xiao et al., 2005). This implies that the broad-spectrum mildew resistance is specified by the RPW8 proteins upstream of SA-dependent signaling.
The RPW8 protein activated resistance is poorly understood. Thus it would be advantageous to provide some insight into the activity and capabilities of the RPW8 protein and use such activity to provide a defense against pathogens at the plasma membrane.